Escherichia coli is the organism of choice for the expression of a wide variety of recombinant proteins for therapeutic, diagnostic and industrial applications.
E. coli generates acetic acid ...(acetate) as an undesirable by-product that has several negative effects on protein production. Various strategies have been developed to limit acetate accumulation or reduce its negative effects to increase the productivity of recombinant proteins. This article reviews recent strategies for reducing or eliminating acetate, including approaches that optimize the protein production process as well as those that involve modifying the host organism itself.
The microbial chiral product (R)-3-hydroxybutyrate (3-HB) is a gateway to several industrial and medical compounds. Acetyl-CoA is the key precursor for 3-HB, and several native pathways compete with ...3-HB production. The principal competing pathway in wild-type Escherichia coli for acetyl-CoA is mediated by citrate synthase (coded by gltA), which directs over 60% of the acetyl-CoA into the tricarboxylic acid cycle. Eliminating citrate synthase activity (deletion of gltA) prevents growth on glucose as the sole carbon source. In this study, an alternative approach is used to generate an increased yield of 3-HB: citrate synthase activity is reduced but not eliminated by targeted substitutions in the chromosomally expressed enzyme. Five E. coli GltA variants were examined for 3-HB production via heterologous overexpression of a thiolase (phaA) and NADPH-dependent acetoacetyl-CoA reductase (phaB) from Cupriavidus necator. In shake flask studies, four variants showed nearly 5-fold greater 3-HB yield compared to the wild-type, although pyruvate accumulated. Overexpression of either native thioesterases TesB or YciA eliminated pyruvate formation, but diverted acetyl-CoA towards acetate formation. Overexpression of pantothenate kinase similarly decreased pyruvate formation but did not improve 3-HB yield. Controlled batch studies at the 1.25 L scale demonstrated that the GltAA267T variant produced the greatest 3-HB titer of 4.9 g/L with a yield of 0.17 g/g. In a phosphate-starved repeated batch process, E. coli ldhA poxB pta-ackA gltA::gltA.sup.A267T generated 15.9 g/L 3-HB (effective concentration of 21.3 g/L with dilution) with yield of 0.16 g/g from glucose as the sole carbon source. This study demonstrates that GltA variants offer a means to affect the generation of acetyl-CoA derived products. This approach should benefit a wide range of acetyl-CoA derived biochemical products in E. coli and other microbes. Enhancing substrate affinity of the introduced pathway genes like thiolase towards acetyl-CoA will likely further increase the flux towards 3-HB while reducing pyruvate and acetate accumulation.
Microbial production of lactic acid Eiteman, Mark A; Ramalingam, Subramanian
Biotechnology letters,
05/2015, Volume:
37, Issue:
5
Journal Article
Peer reviewed
Open access
Lactic acid is an important commodity chemical having a wide range of applications. Microbial production effectively competes with chemical synthesis methods because biochemical synthesis permits the ...generation of either one of the two enantiomers with high optical purity at high yield and titer, a result which is particularly beneficial for the production of poly(lactic acid) polymers having specific properties. The commercial viability of microbial lactic acid production relies on utilization of inexpensive carbon substrates derived from agricultural or waste resources. Therefore, optimal lactic acid formation requires an understanding and engineering of both the competing pathways involved in carbohydrate metabolism, as well as pathways leading to potential by-products which both affect product yield. Recent research leverages those biochemical pathways, while researchers also continue to seek strains with improved tolerance and ability to perform under desirable industrial conditions, for example, of pH and temperature.
Altering metabolic flux at a key branch point in metabolism has commonly been accomplished through gene knockouts or by modulating gene expression. An alternative approach to direct metabolic flux ...preferentially toward a product is decreasing the activity of a key enzyme through protein engineering. In Escherichia coli, pyruvate can accumulate from glucose when carbon flux through the pyruvate dehydrogenase complex is suppressed. Based on this principle, 16 chromosomally expressed AceE variants were constructed in E. coli C and compared for growth rate and pyruvate accumulation using glucose as the sole carbon source. To prevent conversion of pyruvate to other products, the strains also contained deletions in two nonessential pathways: lactate dehydrogenase (
) and pyruvate oxidase (
). The effect of deleting phosphoenolpyruvate synthase (
) on pyruvate assimilation was also examined. The best pyruvate-accumulating strains were examined in controlled batch and continuous processes. In a nitrogen-limited chemostat process at steady-state growth rates of 0.15 to 0.28 h
, an engineered strain expressing the AceEH106V variant accumulated pyruvate at a yield of 0.59 to 0.66 g pyruvate/g glucose with a specific productivity of 0.78 to 0.92 g pyruvate/g cells·h. These results provide proof of concept that pyruvate dehydrogenase complex variants can effectively shift carbon flux away from central carbon metabolism to allow pyruvate accumulation. This approach can potentially be applied to other key enzymes in metabolism to direct carbon toward a biochemical product.
Microbial production of biochemicals from renewable resources has become an efficient and cost-effective alternative to traditional chemical synthesis methods. Metabolic engineering tools are important for optimizing a process to perform at an economically feasible level. This study describes an additional tool to modify central metabolism and direct metabolic flux to a product. We have shown that variants of the pyruvate dehydrogenase complex can direct metabolic flux away from cell growth to increase pyruvate production in Escherichia coli. This approach could be paired with existing strategies to optimize metabolism and create industrially relevant and economically feasible processes.
The biorefinery concept makes use of renewable lignocellulosic biomass to produce commodities sustainably. A synthetic microbial consortium can enable the simultaneous utilization of sugars such as ...glucose and xylose to produce biochemicals, where each consortium member converts one sugar into the target product. In this study, woody biomass was used to generate glucose and xylose after pretreatment with 20% (
w
/
v
) sulfuric acid and 60-min reaction time. We compared several strategies for detoxification with charcoal and sodium borohydride treatments to improve the fermentability of this hydrolysate in a defined medium for the production of the growth-associated product pyruvate. In shake flask culture, the highest pyruvate yield on xylose of 0.8 g/g was found using pH 6 charcoal-treated hydrolysate. In bioreactor studies, a consortium of two engineered
E. coli
strains converted the mixture of glucose and xylose in batch studies to 12.8 ± 2.7 g/L pyruvate in 13 h. These results demonstrate that lignocellulosic biomass as the sole carbon source can be used to produce growth-related products after employing suitable detoxification strategies.
Pyruvic acid (pyruvate) is a cellular metabolite found at the biochemical junction of glycolysis and the tricarboxylic acid cycle. Pyruvate is used in food, cosmetics, pharmaceutical and agricultural ...applications. Microbial production of pyruvate from either yeast or bacteria relies on restricting the natural catabolism of pyruvate, while also limiting the accumulation of the numerous potential by-products. In this review we describe research to improve pyruvate formation which has targeted both strain development and process development. Strain development requires an understanding of carbohydrate metabolism and the many competing enzymes which use pyruvate as a substrate, and it often combines classical mutation/isolation approaches with modern metabolic engineering strategies. Process development requires an understanding of operational modes and their differing effects on microbial growth and product formation.
Citramalic acid (citramalate) serves as a five-carbon precursor for the chemical synthesis of methacrylic acid. We compared citramalate and acetate accumulation from glycerol using
Escherichia coli
...strains expressing a modified citramalate synthase gene
cimA
from
Methanococcus jannaschii
. These studies revealed that
gltA
coding citrate synthase,
leuC
coding 3-isopropylmalate dehydratase, and acetate pathway genes play important roles in elevating citramalate and minimizing acetate formation. Controlled 1.0 L batch experiments confirmed that deletions in all three acetate-production genes (
poxB
,
ackA,
and
pta
) were necessary to reduce acetate formation to less than 1 g/L during citramalate production from 30 g/L glycerol. Fed-batch processes using MEC568/pZE12-
cimA
(
gltA leuC ackA
-
pta poxB
) generated over 31 g/L citramalate and less than 2 g/L acetate from either purified or crude glycerol at yields exceeding 0.50 g citramalate/g glycerol in 132 h. These results hold promise for the viable formation of citramalate from unrefined glycerol.
Limiting an essential nutrient has a profound impact on microbial growth. The notion of growth under limited conditions was first described using simple Monod kinetics proposed in the 1940s. ...Different operational modes (chemostat, fed-batch processes) were soon developed to address questions related to microbial physiology and cell maintenance and to enhance product formation. With more recent developments of metabolic engineering and systems biology, as well as high-throughput approaches, the focus of current engineers and applied microbiologists has shifted from these fundamental biochemical processes. This review draws attention again to nutrient-limited processes. Indeed, the sophisticated gene editing tools not available to pioneers offer the prospect of metabolic engineering strategies which leverage nutrient limited processes. Thus, nutrient- limited processes continue to be very relevant to generate microbially derived biochemicals.
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