Abstract

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.

中文翻译：

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结合产酸克雷伯氏菌KMS004的代谢工程和进化适应，可显着提高低营养培养基中的光学纯D-（-）-乳酸产量和比生产率

摘要

在这项研究中，催娩克氏KMS004（Δ的adhE Δ PTA-ackA）进一步由所述缺失的重新设计frdABCD和PFLB（ - - ） -乳酸产量的基因转移的碳通量通过d。在高葡萄糖浓度的发酵过程中，由于其不足以产生用于生物合成的乙酰辅酶A的能力，所得菌株命名的催产克氏杆菌KIS004显示出较差的生长和葡萄糖消耗。因此，对菌株进行了进化适应，以克服生长受损和乙酸营养缺陷型。演进的催娩克氏KIS004-91T应变表现出更高显著利用葡萄糖率和d - （ - ） -乳酸生产为再生NAD一个主路由⁺。通过该菌株获得浓度为133 g / L（1.48 M）的D-（-）-乳酸，产率和生产率分别为0.98 g / g和2.22 g / L / h。据我们所知，该菌株在其他先前工作中提供了相对较高的比生产率，为1.91 g / gCDW / h。木薯淀粉还用于证明D-（-）-乳酸生产的潜在低成本可再生基质。D-（-）-乳酸的生产成本估计为$ 3.72 / kg。因此，KIS004-91T菌株有可能成为替代的生物催化剂，以工业规模提供更具经济竞争力的D-（-）-乳酸生产。

关键点

•KIS004-91T在低盐培养基中产生的光学纯D-（-）-乳酸盐高达1.48M。

•与其他已报告的菌株相比，它具有最高的D-（-）-乳酸特异性生产力。

•木薯淀粉作为廉价且可再生的底物用于D-（-）-乳酸的生产。

•减少了与培养基，发酵，纯化和废物处理有关的成本。