The outlooks and key challenges in renewable biomass feedstock utilization for value-added platform chemical via bioprocesses.

The conversion of renewable biomass feedstock into value-added products via bioprocessing platforms has become attractive because of environmental and health concerns. Process performance and cost competitiveness are major factors in the bioprocess design to produce desirable products from biomass feedstock. Proper pretreatment allows delignification and hemicellulose removal from the liquid fraction, allowing cellulose to be readily hydrolyzed to monomeric sugars. Several industrial products are produced via sugar fermentation using either naturally isolated or genetically modified microbes. Microbial platforms play an important role in the synthesis of several products, including drop-in chemicals, as-in products, and novel compounds. The key elements in developing a fermentation platform are medium formulation, sterilization, and active cells for inoculation. Downstream bioproduct recovery may seem like a straightforward chemical process, but is more complex, wherein cost competitiveness versus recovery performance becomes a challenge. This review summarizes the prospects for utilizing renewable biomass for bioprocessing.

Genome engineering of Kluyveromyces marxianus for high D-( -)-lactic acid production under low pH conditions.

Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies in cell growth and D-lactate production at high substrate concentrations. Complex nutrients or high cell density were thus required to support growth and D-lactate production with a potential to increase medium and process cost of industrial-scale D-lactate production. As an alternative microbial biocatalyst, a Crabtree-negative and thermotolerant yeast Kluyveromyces marxianus was engineered in this study to produce high titer and yield of D-lactate at a lower pH without growth defects. Only pyruvate decarboxylase 1 (PDC1) gene was replaced by a codon-optimized bacterial D-lactate dehydrogenase (ldhA). Ethanol, glycerol, or acetic acid was not produced by the resulting strain, KMΔpdc1::ldhA. Aeration rate at 1.5 vvm and culture pH 5.0 at 30 °C provided the highest D-lactate titer of 42.97 ± 0.48 g/L from glucose. Yield and productivity of D-lactate, and glucose-consumption rate were 0.85 ± 0.01 g/g, 0.90 ± 0.01 g/(L·h), and 1.06 ± 0.00 g/(L·h), respectively. Surprisingly, D-lactate titer, productivity, and glucose-consumption rate of 52.29 ± 0.68 g/L, 1.38 ± 0.05 g/(L·h), and 1.22 ± 0.00 g/(L·h), respectively, were higher at 42 °C compared to 30 °C. Sugarcane molasses, a low-value carbon, led to the highest D-lactate titer and yield of 66.26 ± 0.81 g/L and 0.91 ± 0.01 g/g, respectively, in a medium without additional nutrients. This study is a pioneer work of engineering K. marxianus to produce D-lactate at the yield approaching theoretical maximum using simple batch process. Our results support the potential of an engineered K. marxianus for D-lactate production on an industrial scale. KEY POINTS: • K. marxianus was engineered by deleting PDC1 and expressing codon-optimized D-ldhA. • The strain allowed high D-lactate titer and yield under pH ranging from 3.5 to 5.0. • The strain produced 66 g/L D-lactate at 30 °C from molasses without any additional nutrients.

Synthesis of Nicotinamide Mononucleotide from Xylose via Coupling Engineered Escherichia coli and a Biocatalytic Cascade.

ß-Nicotinamide mononucleotide (NMN) has recently gained attention for a nutritional supplement because it is an intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD+ ). In this study, we developed NMN synthesis by coupling two modules. The first module is to culture E. coli MG1655 ▵tktA ▵tktB ▵ptsG to metabolize xylose to generate D-ribose in the medium. The supernatant containing D-ribose was applied in the second module which is composed of EcRbsK-EcPRPS-CpNAMPT reaction to synthesize NMN, that requires additional enzymes of CHU0107 and EcPPase to remove feedback inhibitors ADP and pyrophosphate. The second module can be rapidly optimized by comparing NMN production determined by the cyanide assay. Finally, 10 mL optimal biocascade reaction generated NMN with a good yield of 84 % from 1 mM D-ribose supplied from the supernatant of E. coli MG1655 ▵tktA ▵tktB ▵ptsG. Our results can further guide researchers to metabolically engineer E. coli for NMN synthesis.

Combining metabolic engineering and evolutionary adaptation in Klebsiella oxytoca KMS004 to significantly improve optically pure D-(-)-lactic acid yield and specific productivity in low nutrient medium.

In this study, K. oxytoca KMS004 (ΔadhE Δpta-ackA) was further reengineered by the deletion of frdABCD and pflB genes to divert carbon flux through D-(-)-lactate production. During fermentation of high glucose concentration, the resulted strain named K. oxytoca KIS004 showed poor in growth and glucose consumption due to its insufficient capacity to generate acetyl-CoA for biosynthesis. Evolutionary adaptation was thus employed with the strain to overcome impaired growth and acetate auxotroph. The evolved K. oxytoca KIS004-91T strain exhibited significantly higher glucose-utilizing rate and D-(-)-lactate production as a primary route to regenerate NAD+. D-(-)-lactate at concentration of 133 g/L (1.48 M), with yield and productivity of 0.98 g/g and 2.22 g/L/h, respectively, was obtained by the strain. To the best of our knowledge, this strain provided a relatively high specific productivity of 1.91 g/gCDW/h among those of other previous works. Cassava starch was also used to demonstrate a potential low-cost renewable substrate for D-(-)-lactate production. Production cost of D-(-)-lactate was estimated at $3.72/kg. Therefore, it is possible for the KIS004-91T strain to be an alternative biocatalyst offering a more economically competitive D-(-)-lactate production on an industrial scale. KEY POINTS: • KIS004-91T produced optically pure D-(-)-lactate up to 1.48 M in a low salts medium. • It possessed the highest specific D-(-)-lactate productivity than other reported strains. • Cassava starch as a cheap and renewable substrate was used for D-(-)-lactate production. • Costs related to media, fermentation, purification, and waste disposal were reduced.

Re-engineering Escherichia coli KJ122 to enhance the utilization of xylose and xylose/glucose mixture for efficient succinate production in mineral salt medium.

Escherichia coli KJ122 was previously engineered to produce high concentration and yield of succinate in mineral salt medium containing glucose and sucrose under anaerobic conditions. However, this strain does not efficiently utilize xylose. To improve the xylose uptake and utilization in the strain KJ122, xylFGH and xylE genes were individually and simultaneously deleted. E. coli KJ12201 (KJ122::ΔxylFGH) exhibited superior abilities in growth, xylose consumption, and succinate production compared to those of the parental strain KJ122. However, E. coli KJ12202 (KJ122::ΔxylE) lessened xylose consumption due to an ATP deficit for metabolizing xylose thus making succinate production from xylose not preferable. Moreover, E. coli KJ12203 (KJ122::ΔxylFGHΔxylE) exhibited an impaired growth on xylose due to lacking of xylose transporters. After performing metabolic evolution, the evolved KJ12201-14T strain exhibited a great improvement in succinate production from pure xylose with higher concentration and productivity about 18 and 21%, respectively, compared to KJ12201 strain. During fed-batch fermentation, KJ12201-14T also produced succinate from xylose at a concentration, yield, and overall productivity of 84.6 ± 0.7 g/L, 0.86 ± 0.01 g/g and 1.01 ± 0.01 g/L/h, respectively. KJ12201 and KJ12201-14T strains co-utilized glucose/xylose mixture without catabolite repression. Both strains produced succinate from glucose/xylose mixture at concentration, yield, and overall and specific productivities of about 85 g/L, 0.85 g/g, 0.70 g/L/h, and 0.44 g/gCDW/h, respectively. Based on our results, KJ12201 and KJ12201-14T strains exhibited a greater performance in succinate production from xylose containing medium than those of other published works. They would be potential strains for the economic bio-based succinate production from xylose.

Efficient reduction of the formation of by-products and improvement of production yield of 2,3-butanediol by a combined deletion of alcohol dehydrogenase, acetate kinase-phosphotransacetylase, and lactate dehydrogenase genes in metabolically engineered Klebsiella oxytoca in mineral salts medium.

Klebsiella oxytoca KMS005 (∆adhE∆ackA-pta∆ldhA) was metabolically engineered to improve 2,3-butanediol (BDO) yield. Elimination of alcohol dehydrogenase E (adhE), acetate kinase A-phosphotransacetylase (ackA-pta), and lactate dehydrogenase A (ldhA) enzymes allowed BDO production as a primary pathway for NADH re-oxidation, and significantly reduced by-products. KMS005 was screened for the efficient glucose utilization by metabolic evolution. KMS005-73T improved BDO production at a concentration of 23.5±0.5 g/L with yield of 0.46±0.02 g/g in mineral salts medium containing 50 g/L glucose in a shake flask. KMS005-73T also exhibited BDO yields of about 0.40-0.42 g/g from sugarcane molasses, cassava starch, and maltodextrin. During fed-batch fermentation, KMS005-73T produced BDO at a concentration, yield, and overall and specific productivities of 117.4±4.5 g/L, 0.49±0.02 g/g, 1.20±0.05 g/Lh, and 27.2±1.1 g/gCDW, respectively. No acetoin, lactate, and formate were detected, and only trace amounts of acetate and ethanol were formed. The strain also produced the least by-products and the highest BDO yield among other Klebsiella strains previously developed.